The disease tuberculosis has become a major health problem nationally and internationally. More people die of infections with M. tuberculosis worldwide than any other infectious agent. Central to the disease process in tuberculosis are the interactions of the bacilli with host macrophages. The infection of macrophages by M. tuberculosis can be divided into three steps: adherence, entry, and intracellular survival. The role virulence factors of M. tuberculosis play in these complex interactions is virtually unknown. The goal of this proposed fellowship is to identify, clone and characterize genes of M. tuberculosis which are required for uptake, intracellular survival, and multiplication within human macrophages. Mycobacterium smegmatis will be used to clone these potential virulence factor genes of M tuberculosis. M. smegmatis is an ideal strain to use in these studies because it grows rapidly in the laboratory (in days instead of weeks as for M. tuberculosis), readily expresses genes from other mycobacteria, can be genetically manipulated by various techniques, is nonpathogenic, and is internalized and killed by macrophages. The specific aims are to: 1. Prepare a genomic library from a virulent strain of M. tuberculosis by using a plasmid vector system. Mycobacteria smegmatis will be transformed with the genomic library by electroporation. Antibiotic resistant transformations will be selected and pooled. 2. M. smegmatis transformants will be used to infect monolayers of human macrophages and those clones which invade and/or survive intracellularly within macrophages will be isolated. 3. Plasmids from M. smegmatis clones recovered from macrophage monolayers will be isolated, restriction mapped, and sequenced to characterize the cloned gene(s). 4. The role of these cloned genes in uptake and intracellular survival of M. tuberculosis within macrophages will be verified by construction of mutations in these gene(s). The mutations will be introduced into the chromosome of virulent Mycobacterium tuberculosis by gene replacement techniques. These mutant strains will then be tested for their abilities to enter and survive within macrophages as compared to the virulent wild- type strain.